Characterization of the CAT2 transporter from Solanum lycopersicum (Tomato).

The vacuole is the largest organelle in plant cells and it is essential for life. Although the vacuole has always been associated with turgor regulation and storage of water and metabolites, it is now clear that this organelle is also involved in pH homeostasis, cell energetics and signal transduction. Among metabolites exchanged in vacuoles, amino acids are particularly represented since they play both metabolic and signaling functions. In particular Arginine is crucial for plant cells, being a precursor for nitric oxide and polyamine. We have very recently expressed the CAT2 transporter from Solanum lycopersicum in E.coli. The recombinant protein has been reconstituted in the artificial membrane of proteoliposomes and the transporter has been functionally characterized. The ability for CAT2 to transport cationic amino acids has been demonstrated measuring the uptake of several radiolabeled amino acids in proteoliposomes. Arginine, Lysine and the nonproteogenic amino acid Ornithine are the most efficiently transported. Arginine transport is stimulated by the presence of cholesterol in the membrane. Since the physiological pH of vacuoles is acidic inside, the transport activity has been recorded as a function of the pH gradient. Optimal transport was observed in the presence of intraliposomal pH 5.5 and external pH 8.0-8.5. The possible mechanism of pH control has been predicted on the basis of the homology model of CAT2 obtained using the 3D structure of the ApcT transporter from Geobacillus Kaustophilus as a template. Glu 142 has been hypothesized of being involved in proton binding. Since the existence of a non-neuronal cholinergic system has also been ascertained in plants, the possible role of CAT2 in Acetylcholine transport in the vacuoles has been investigated. Acetylcholine transport mediated by CAT2 has been revealed. This data suggests that CAT2 can promote the Acetylcholine storage into the vacuole.